Hereditary pulmonary alveolar proteinosis (hPAP) is a disorder of increased surfactant accumulation resulting in respiratory failure for which no pharmacologic therapy exists. It is caused by disruption of GM-CSF signaling to alveolar macrophages (AMs) by mutations in CSF2RA or CSF2RB, which encode GM-CSF receptor (GM-R) and subunits, respectively. The long-term goal is to develop gene therapy to restore GM-CSF signaling in AMs and, thereby, GM-CSF-dependent AM functions critical to surfactant clearance, alveolar homeostasis, lung function and host defense. The objective here is to evaluate a novel therapeutic approach, pulmonary macrophage transplantation (PMT). Preliminary data show that GM-R deficient (GM-RKO) mice develop hPAP lung disease that is identical to hPAP in humans including an increased level of pulmonary GM-CSF, which confers a selective survival advantage to AMs with functional GM-Rs. The central hypothesis is that safety-enhanced, lentiviral vector-mediated GM-R expression in hematopoietic stem/precursor cells (HSPCs), expansion into macrophages, and autologous PMT of gene-corrected cells without myeloablation will be effective and safe as therapy of hPAP. Preliminary data show that a single PMT treatment can correct hPAP in GM-RKO mice for at least one year (the longest time evaluated) without associated adverse events, and that human HSPCs can be readily transduced using well-established methods in the applicants' laboratory and expanded into macrophages expressing functional GM-Rs. The rationale is that anticipated results will inform the design of future a clinical trial and provide the preclinical safety and efficacy data, and GMP manufacturing procedures and validation data required to obtain regulatory approval to test this approach in humans. The hypothesis will be tested in four specific aims: 1) determine the efficiency and kinetics of gene transfer/PMT therapy of hPAP in GM-RKO mice; 2) optimize the expansion of human HSPC-derived, gene- corrected macrophages with maximum PMT engraftment potential; 3) determine the safety of gene transfer/PMT in preclinical studies related to gene transfer, macrophage expansion from HSPCs, PMT, and pharmacologic depletion of transduced cells in GM-RKO mice; 4) develop and validate protocols for the manufacture of HSPC-derived, gene-corrected macrophages, and write a clinical protocol and investigational new drug application for gene transfer/PMT therapy of hPAP. The approach is innovative because it departs markedly from the current inefficient, highly invasive method of physically removing surfactant by whole lung lavage and in- stead uses a novel approach to restore AM function. The proposed research is significant because it is expected to establish the feasibility of a specific therapy for children with hPAP and a new type of therapy (PMT) that may be useful for other diseases, and evaluate multiple safety improvements to reduce the risks of gene therapy. Results will inform a fundamental mechanism by which GM-CSF regulates AM population size, pro- vide an estimate of AM lifespan, and lay the foundation for the development of macrophage-based therapy.